Annex

Annex A: Amino acid sequence alignment of methyltransferase-like proteins

Comparison of predicted amino acid sequences of rRNA dimethylases and related mitochondrial transcription factors. Shaded positions are conserved in 40 % (light grey), 70 % (grey) or 100 % (black) of aligned sequences. Regions corresponding to eight conserved sequence motifs of SAM-dependent methyltransferases that are involved in SAM binding (motifs I-III) and in RNA binding and catalysis (motifs IV-VIII) are indicated. Grey bars below mark alignment sections that were included to reconstruct methyltransferase phylogeny. Sequences were aligned of AtMetA (At5g66360), AtMetB (At2g47420) and Pfc1 (At1g01860) from Arabidopsis thaliana, PtMetA (fgenesh1_pg.C_LG_XVIII000509), PtMetB (eugene3.00140665) and PtPfc1 (eugene3.00140230) from Populus trichocarpa, MtMetA (AC136451.9) and MtMetB (CA922412 and BF632918) from Medicago truncatula, GmMetB (CX703076.1) from Glycine max., LeMetB (BT013951.1) from Lycopersicon esculentum, OsMetA (XP_478337.1B) and OsMetB (AK099711.1) from Oryza sativa, ZmMetA (AY108752.1) and ZmMetB (CO520188.1 and CG266357.1) from Zea mays, sc-mtTFB (NP_013955.1) and sc-Dim1 (NP_015057.1) from Saccharomyces cerevisiae, kl-mtTFB (P87250) and kl-Dim1 (XP_453098.1) from Kluyveromyces lactis, sk-mtTFB (P87292) from Saccharomyces kluyveri, sp-mtTFB (CAB65608.1) from Schizosaccharomyces pombe, h-mtTFB1 (NP_057104.1), h-mtTFB2 (NP_071761.1) and h-Dim (AAH10874.1) from Homo sapiens, d-mtTFB1 (CG7319-PA), d-mtTFB2 (CG3910-PA) and d-Dim (NP_651660.1) from Drosophila melanogaster, m-mtTFB1 (NP_666186.1) and m-mtTFB2 (NP_032275.1) from Mus musculus, r-mtTFB1 (NP_852139.2) and r-mtTFB2 (NP_001008294.1) from Rattus norvegicus, x-mtTFB (AAH56010.1) from Xenopus laevis, EcKsgA (P06992) from Escherichia coli, PaeKsgA (H83571) from Pseudomonas aeruginosa and BcErmC (P13956) from Bacillus subtilis. Amino acid sequences were derived from genomic and cDNA sequences using the FEX tool at www.softberry.com and the Translate tool at www.expasy.ch.

Annex B: Predicted subcellular targeting of plant methyltransferase-like proteins

Predictions of targeting to the mitochondrion (M), nucleus (N), or plastid (P) by the computer algorithms listed in Table 11 are documented; Mitoprot predictions are given as probabilities of mitochondrial targeting. Proteins are designated as in Annex A.

Protein

Predotar

TargetP

Mitoprot

Psort

iPsort

AtMetA

Possibly M

M

0.59

M or N

M

AtMetB

Not M or P

Not M or P

0,25

N

Not M or P

AtPfc1

P

P

0.24

Not M or P

P

PtMetA

Possibly M

M

0.99

N

M

PtMetB

Not M or P

Not M or P

0.47

Not M or P

M

PtPfc1

Possibly P

P

0.79

P or N

P

MtMetA

M

M

0.93

M

Not M or P

MtMetB

Not M or P

Not M or P

0.09

Not M or P

Not M or P

GmMetB

Not M or P

Not M or P

0.23

Not M or P

Not M or P

LeMetB

Not M or P

Not M or P

0.23

Not M or P

M

OsMetA

M

M

0.78

M

M

OsMetB

Not M or P

Not M or P

0.16

Not M or P

Not M or P

ZmMetB

Not M or P

Not M or P

0.41

Not M or P

Not M or P

ZmMetA

M

M

1.0

M

M

Annex C: Threaded structural models of Arabidopsis RpoTm and RpoTmp

The RpoTm and RpoTmp structural models (right) are based on T7 RNA polymerase crystal structures (Cheetham, et al., 1999); (Cheetham and Steitz, 1999). The N-terminal domain of the T7 enzyme is coloured pale yellow; the thumb, palm and fingers subdomains are shown in pale green, pale red and pale blue, respectively; the specificity loop (arrow) is bright blue and the double helix of the DNA template is orange (compare I.3.2.2 and Figure 5). Corresponding regions of the RpoTm and RpoTmp structural models are coloured accordingly. The RpoTm and RpoTmp models exclude the first 104 (RpoTm) and 150 (RpoTmp) amino acids from the N-terminus, which are not conserved with the T7 RNA polymerase. The secondary structure of regions corresponding to the T7 enzyme specificity loop (arrows) is unresolved in the RpoTm and RpoTmp models. The coordinates of the threaded models of RpoTm and RpoTmp were derived from www.bmm.icnet.uk/servers/3djigsaw/ (Bates, et al., 2001;Bates and Sternberg, 1999;Contreras-Moreira and Bates, 2002) with the program choosing the T7 RNA polymerase/promoter DNA complex [PDB number 1cez (Cheetham, et al., 1999)] and the structure of a transcribing T7 RNA polymerase initiation complex [PDB number 1qln (Cheetham and Steitz, 1999)] as the starting structures to create the threaded model coordinates for RpoTm and RpoTmp, respectively. The images were generated using PyMol v0.98 (http://delsci.com/rel/0_98/). Choosing the T7 enzyme/promoter DNA complex (PDB number 1cez) as structure template and providing the RNA polymerase alignment shown in Annex A, SWISS-MODEL [http://swissmodel.expasy.org/, (Guex and Peitsch, 1997;Peitsch, 1995;Schwede, et al., 2003)] was independently used to build 3D models of RpoTm and RpoTmp polypeptide regions corresponding to thumb, palm and fingers subdomains of the T7 enzyme; the final coordinates generated were highly similar (data not shown).

Annex D: Accession numbers of RNA polymerase sequences

Accession numbers of amino acid sequences of bacteriophage and phage-type RNA polymerases included in the alignment shown in Fig. 32 and Annex E. Protein targeting to the mitochondrion (M) and/or plastid (P) is indicated.

Protein

Accession number

Source

Targeting

At-RpoTm

CAA69331

Arabidopsis thaliana

M

At-RpoTmp

CAC17120

Arabidopsis thaliana

M, P

At-RpoTp

CAA70210

Arabidopsis thaliana

P

Ns-RpoTm

CAC95019

Nicotiana sylvestris

M

Ns-RpoTmp

Q8VWF8

Nicotiana sylvestris

M, P

Ns-RpoTp

P69242

Nicotiana sylvestris

P

Ta-RpoTm

AAF3249

Triticum aestivum

M

Ta-RpoTp

AAB01085

Triticum aestivum

P

Zm-RpoTm

AAD22976

Zea mays

M

Zm-RpoTp

AAD22977

Zea mays

P

sc-Rpo41

P13433

Saccharomyces cerevisiae

M

sp-Rpo41

CAB16197

Schizosaccharomyces pombe

M

h-mtPol

AAC06147

Homo sapiens

M

T7

NP_041960

Bacteriophage T7

-

T3

NP_523301

Bacteriophage T3

-

phiYeO3-12

NP_052071

Bacteriophage phiYeO3-12

-

SP6

P06221

Bacteriophage SP6

-

Annex E: T7 phage and phage-type RNA polymerase sequence alignment

Comparison of amino acid sequences of RpoTm, RpoTmp and RpoTp from Arabidopsis thaliana, Rpo41 from Schizosaccharomyces pombe, the mitochondrial RNA polymerase from Homo sapiens, and the T7 bacteriophage RNA polymerase (see Annex D for accession numbers). Shaded positions are conserved in 60 % (light grey), 80 % (grey) or 100 % (black) of aligned sequences. N-terminal portions of organellar enzymes, which do not align to the T7 sequence, are not displayed. Amino acid sequences were compared using the Multalin algorithm (Corpet, 1988); the alignment was refined according to a secondary structure-based alignment of RpoTm to the T7 sequence generated using the GenTHREADER tool at http://bioinf.cs.ucl.ac.uk/psipred/ (Jones, 1999;McGuffin, et al., 2000;McGuffin and Jones, 2003).


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